JP6700197B2 - Method for purifying protein using caprylic acid - Google Patents

Description

  Protein purification with virus inactivation or removal is performed with caprylic acid.

  The use of biopharmaceuticals or pharmaceutical compositions containing therapeutic proteins to treat diseases, disorders, or conditions is a strategic component of many pharmaceutical and biotechnology companies. Many proteins used in biopharmaceutical compositions are obtained by recombinant production using mammalian cell lines or by purification of body fluids. However, therapeutic proteins produced by such methods may be contaminated with viruses, apparently infectious pathogenic viruses, and may be detrimental to the health of the individual. It is necessary to treat the medium containing the therapeutic protein to remove any potential viral activity.

  Inactivation of infectious pathogenic viruses includes heat inactivation, solvent/detergent (S/D) inactivation, pH inactivation, chemical inactivation, and/or radiation inactivation. S/D inactivation is probably the most widely accepted real-world method that can be done by.

  Most human pathogens are enveloped viruses. They are susceptible to membrane disruption by solvents and surfactants. Inactivation methods using heat, acidic pH, chemicals, and irradiation are harsh and/or invasive and tend to denature or otherwise inactivate the purified therapeutic protein. Therefore, these means have a problem.

  Due to viral inactivation, as part of viral clearance during purification of pharmaceutical products, enveloped viruses are usually inactivated by detergents or low pH treatment steps, during which the pharmaceutical product's Surfactant is added to the solution or the pH is adjusted to approximately 3.7 (3.5-3.9) for up to 6 hours or more. However, detergents are not always effective against all types of enveloped viruses, there may be environmental problems, and with respect to low pH inactivation, at pH higher than 3.8, it becomes ineffective. The window of operation is very narrow because the activation effect is weakened. Conversely, pH values below 3.5 dramatically increase the risk of aggregate formation or denaturation for many proteins.

  EP 0 374 625 shows that caprylic acid has a short-term effect on most enveloped viruses. US 4,939,176 reports a method for inactivating viruses in a solution of biologically active protein by contacting it with caprylic acid. The preferred conditions described for the process were pH 4 to pH 8 and 0.07 (%w/w) to 0.001 (%w/w) non-ionized form of caprylic acid.

  Other methods of viral inactivation by the use of chemicals are known. US 4,540,573 teaches the use of dialkyl phosphates or trialkyl phosphates as antiviral agents. US 4,534,972 describes a method of rendering a solution of therapeutically or immunologically active protein substantially free of infectious agents. In US 4,534,972, a solution of a protein is contacted with a transition metal complex, such as copper phenanthroline, and a reducing agent to effect viral inactivation without substantially affecting the activity of the protein.

  US 5,164,487 relates to the use of caprylic acid for the manufacture of IgG preparations free of aggregation, vasoactive substances and proteolytic enzymes. The method involves contacting a starting material containing IgG with 0.4% to 1.5% caprylic acid followed by chromatographic purification using an ion exchange or hydrophobic matrix.

  Steinbuch et al. showed the use of caprylic acid to precipitate most proteins (other than immunoglobulins) and lipoproteins present in corn ethanol fraction III (Steinbuch et al., 1973). A two-step purification of immunoglobulins from mammalian serum and ascites has been described (McKinney et al., 1987). First, caprylic acid was used to precipitate albumin and other non-IgG proteins, and then ammonium sulfate was added to the supernatant to precipitate IgG.

  During the preparation of human immunoglobulins, caprylic acid is generally recognized as an effective precipitant for most plasma proteins at pH 4.8, as long as parameters such as temperature and ionic strength are optimized. Steinbuch et al. (1969) describe the precipitation of most plasma proteins with caprylic acid without affecting IgG, ceruloplasmin, and IgA. Steinbuch et al. isolated IgG from mammalian serum using caprylic acid and reported that extensive non-immunoglobulin precipitation was best achieved at pHs that were slightly acidic but not below pH 4.5. Plasma was diluted 2:1 with 0.06 M acetate buffer (pH 4.8) and then treated with 2.5 wt% caprylate to initiate precipitation.

EP 0 374 625 US 4,939,176 US 4,540,573 US 4,534,972 US 5,164,487

Steinbuch et al., 1973 McKinney et al., 1987 Steinbuch et al., 1969

  The present invention is particularly useful for inactivating viruses without discontinuous processes and without the need for operator's attention when pH is adjusted automatically by elution in a buffer. Finally, the incorporation of caprylic acid treatment as part of the chromatographic step was accomplished.

The present invention is
i. Loading the stationary phase with a protein-containing sample such that the target protein is bound to said stationary phase;
ii. exposing the solid phase with bound target protein to a solution of caprylic acid at a pH such that the solution of caprylic acid produces free caprylic acid, wherein the solution of caprylic acid is A caprylic acid buffer solution having a measured concentration of 1 to 50 mM;
iii. A method for purifying a target protein from a sample is provided, which comprises a step of eluting the target protein.

  The present invention is particularly useful for inactivating viruses without discontinuous processes and without the need for operator's attention when pH is adjusted automatically by elution in a buffer. Finally, the incorporation of caprylic acid treatment as part of the chromatographic step was accomplished.

One aspect of the present invention is
i. loading the stationary phase with a protein-containing sample so that the target protein is bound to the stationary phase;
ii. exposing the solid phase to a caprylic acid solution; and
iii. A method for purifying a target protein from a sample, which comprises the step of eluting the target protein.

  Further aspects of the invention are: i. loading the sample onto a stationary phase such that the target protein is bound to said stationary phase; ii. exposing the solid phase to a solution of caprylic acid to inactivate the virus , A step of washing away; and iii. a method for inactivating a virus in a protein-containing mixture comprising the step of eluting the target protein.

  Another aspect of the invention is: i. loading the sample onto a stationary phase so that the target protein is bound to said stationary phase; ii. subjecting the solid phase to a caprylic acid solution wash to inactivate the virus And rinsing; and iii. a method for preparing a virus-free protein solution, which comprises the step of eluting the target protein.

  An alternative embodiment of the invention is directed to a method of virus inactivation, wherein a) a sample containing a target protein and b) one or more virus types is loaded onto a stationary phase and washed with a caprylic acid solution. ..

  i. loading the sample onto the stationary phase to allow the target protein to bind to the solid phase; ii. subjecting the solid phase to a caprylic acid solution wash to inactivate and wash away the virus And iii. A method of inactivating a virus comprising a lipid coating, comprising the step of eluting the target protein.

  Further, the present invention includes the steps of: i. loading the sample onto the stationary phase so that the target protein is bound to the solid phase; ii. subjecting the solid phase to a caprylic acid solution wash to inactivate the virus; Rinsing away; and iii. A protein sample essentially free of virus with lipid coating, obtained by a method comprising the step of eluting the target protein.

  Further aspects of the invention are: i. loading the sample onto a stationary phase such that the target protein is bound to the solid phase; ii. exposing the solid phase to a caprylic acid solution to inactivate mycoplasma , Rinsing away; and iii. a method for inactivating mycoplasma in a protein-containing mixture comprising elution of said target protein.

  The invention can also solve further problems that are apparent from the disclosure of the exemplary embodiments.

  One aspect of the invention is directed to the simultaneous viral inactivation and protein purification during the chromatography step. The method of the present invention eliminates the need for technician intervention for the inactivation step and separate column loading. A single passivation and capture step can be performed without technician intervention.

  Typically, purification of the target protein involves inactivating potential viruses in the sample. Accordingly, one aspect of the invention is directed to the preparation of virus-free protein solutions, and virus-free protein solutions prepared according to the methods of the invention. The method of the invention involves viral inactivation by washing the chromatography column or membrane with a caprylic acid-containing wash solvent after loading the protein-containing solution and prior to elution of the protein. .. The method of the present invention also inactivates mycoplasma resulting in a high degree of reduction of Host Cell Protein (HCP).

For protein purification and viral inactivation of the present invention, the caprylic acid solution must contain caprylic acid as a free or non-ionized acid, ie, substantially as free or non-ionized caprylic acid. .. The term "free caprylic acid" and "non-ionized caprylic acid" undissociated form, i.e. protonated form, i.e. to mean a caprylic acid as _{CH 3 (CH 2) 6 CO} 2 H are contemplated.

  The invention is any medium or sample containing one or more target proteins, typically one target protein, which protein may further comprise one or more viruses or potentially Intended for separation or purification from a medium or sample containing or containing The sample may be crude medium or may be partially washed, filtered, diafiltered, or partially purified by any method. Suitably, the sample is selected from the group consisting of fermentation broth, cell culture, cell line medium, ascites, tissue culture medium, transgenic cell medium, human or animal blood, human or animal plasma including plasma fraction. It Typically, the sample is selected from cell line media such as transgenic cell media and human or animal plasma.

  A sample or protein-containing mixture is a fluid containing proteins, typically proteins with biological or therapeutic activity. The fluid can be any fluid that can be contaminated by viruses. Non-limiting examples of fluids include cell lysates (lysates), cell supernatants, placenta extracts, or culture extracts of tissues and cells such as ascites; blood, plasma, serum, milk, saliva, semen, etc. Body fluid; or any other fluid containing active protein, or any purified, partially purified, or unpurified (crude) thereof, including the eluate from the immediately preceding purification step. Includes fluids or colloids.

  As mentioned, the samples used in the present invention have cell line media such as transgenic cell media and human or animal plasma as their sources.

  A sample of the invention having a protein of the invention may be a cell that naturally produces the protein, from an organism that is genetically engineered to express the protein, or recombinantly produces the protein. Can be obtained from the stock. Non-limiting examples of transgenic organisms include those organisms disclosed herein that have been genetically engineered to express a protein of interest. The sample of the invention containing the protein may be from an organism or a transgenic organism and may be extracted from body fluids, tissues or organs, or other sources of organism origin, using routine methods well known to those skilled in the art. May be obtained from Furthermore, various prokaryotic and/or eukaryotic expression systems, such as for recombinantly expressing the proteins disclosed herein, can also be sources of the samples of the invention. The expression system as a source of the protein of the present invention includes inducible expression, non-inducible expression, constitutive expression, tissue-specific expression, cell-specific expression, virus-mediated expression, stable integration (stably-integrated). Expression and transient expression are included without limitation.

  The protein of the present invention may be encoded by a polynucleotide cloned into an expression vector. Prokaryotic, eukaryotic, yeast, insect, mammalian and other suitable expression vectors are well known to those of skill in the art and are commercially available.

  After recovery from the initial organism, transgenic organism, or cell culture system, the sample containing the protein of the invention may undergo a purification or filtration process, including a diafiltration process. This initial or preliminary sample purification may include one or more of concentration of proteins in the sample, intermediate purification steps to remove impurities, and final purification (polishing) to remove additional impurities and protein variants. Good.

The object of the invention is the inactivation of viruses in protein-containing samples. The phrase "virus inactivation" is a reduction ("reduction") in the number of viral particles in a particular sample and/or the infectivity and/or the ability of viral particles in a particular sample to replicate. Is intended to mean, but is not limited to, decreased activity. The term "viral inactivation" means rendering a virion that is normally capable of replication in a state in which it is unable to replicate. The term further encompasses at least partial removal of the virus from the sample, or a reduction or reduction in the number thereof. The term encompasses performing the method when the presence or absence of the virus is unknown. For example, for proteins derived from whole blood or plasma or cell supernatant, the presence or absence of virus has not been characterized and there is no guarantee that the plasma and cell supernatant derived proteins will be safe to administer to an individual. The method may be applied. Embodiments of the present invention provide, but are not limited to, a reduction in viral load of greater than 6 log ₁₀ . That is, the method results in a reduction in viral replication of 1,000,000 compared to controls, based on current detection limits.

  The decrease in the number and/or activity of said viral particles is from about 1% to about 100%, preferably about 20% to about 100%, more preferably about 30% to about 100%, more preferably about 40% to about. 100%, even more preferably about 50% to about 100%, even more preferably about 60% to about 100%, even more preferably about at least about 70%, such as at least about 80%, more typically at least about It can be of the order of 90%, for example about 95% to 100%, preferably about 95% to about 100%, typically about 99 to about 100%.

  In one non-limiting embodiment, the total amount of virus in the purified antibody product is the ID50 (amount of virus that infects 50% of the target population of virus) or PFU (plaque formation) for that virus, if present. Units), preferably at least 100 times less than the ID50/PFU for the virus, more preferably at least 10,000 times less than the ID50/PFU for the virus, and even more preferably at least the ID50/PFU for the virus. 1,000,000 times less.

  As will be appreciated by those in the art, the method of the invention can inactivate the virus in the sample and subsequently remove it from the sample, or the method can either inactivate the virus and remove it from the sample. You can do both.

  Aspects herein disclose, in part, a virus that includes a lipid coating. Complete viral particles, known as virions, consist of nucleic acids, either DNA or RNA, and are surrounded by a protective coating of proteins called capsids. Viruses can be classified into non-enveloped and enveloped viruses. As used herein, the term “virus comprising a lipid coating” refers to any virus that includes a lipid-containing membrane or envelope, eg, an enveloped virus. In the enveloped virus, the capsid is wrapped in the lipoprotein membrane or envelope. This envelope is derived from the host cell and consists mostly of lipids that are not encoded by the viral genome, as the virus "buries" from the surface of the host cell. Envelope viruses range in size from about 45-55 nm to about 120-200 nm. Viruses containing lipid coatings capable of infecting mammalian cells include DNA viruses such as herpesviridae, poxviridae, or hepadnaviridae; flaviviridae, togaviridae viruses. RNA viruses such as viruses, viruses of the Coronavirus family, viruses of the Deltavirus family, viruses of the Orthomyxoviridae family, viruses of the Paramyxoviridae family, viruses of the Rhabdoviridae family, viruses of the Bunyaviridae family, or viruses of the Filoviridae family; And a reverse transcription virus such as a virus of the retroviridae or a virus of the hepadnaviridae. Non-limiting examples of viruses that include lipid coatings include human immunodeficiency virus, Sindbis virus, herpes simplex virus, pseudorabies virus, Sendai virus, vesicular stomatitis virus, West Nile virus, bovine viral diarrhea virus, corona. Virus, equine arthritis virus, severe acute respiratory syndrome virus, murine leukemia virus (MuLV), infectious bovine rhinotracheitis virus (IBRV), or vaccinia virus.

  Non-enveloped virus refers to a virus whose capsid has no lipoprotein membrane or envelope. In non-enveloped viruses, the capsid mediates attachment to host cells and entry into the interior. Capsids are generally either helical or icosahedral. Non-enveloped viruses range in size from about 18-26 nm to about 70-90 nm. Non-enveloped viruses that can infect mammalian cells include, for example, parvoviridae, adenoviridae, birnaviridae, papillomaviridae, polyomaviridae, picornavirus. Family viruses, reoviridae viruses, and caliciviridae viruses.

  The virus may be, but is not limited to, MuLV, IBRV, HIV, Hepatitis B, Hepatitis C, Ebola, West Nile, and Hantavirus, including but not limited to transfusion blood and blood-based products. Has the ability to contaminate. It is difficult to remove or inactivate viral factors potentially present in blood products without significantly sacrificing protein quality and/or functionality. Non-limiting examples of industrially relevant viruses include Parvoviridae, Paramyxoviridae, Orthomyxoviridae, Bunyaviridae, Rhabdoviridae, Reoviridae, Togaviridae, Caliciviridae, The Reoviridae and the Picornaviridae.

  Viruses include, but are not limited to, single-stranded DNA viruses, double-stranded DNA viruses, double-stranded RNA viruses, and single-stranded RNA viruses. In a further aspect, the virus is an enveloped virus. Envelope viruses include, but are not limited to, retroviruses, herpesviruses, or hepadnaviruses. In all variants of the invention, the virus is adenovirus, African swine fever strain virus, arenavirus, arterivirus, astrovirus, baculovirus, budnavirus, barnavirus, birnavirus, bromovirus, bunyavirus, calicivirus, mosquito virus. Pirovirus, Carla virus, Calimovirus, Circovirus, Closterovirus, Comovirus, Coronavirus, Cotricovirus (cotricovirus), Cystovirus, Deltavirus, Dianthovirus (dianthovirus), Enamovirus, Filovirus, Flavivirus, Phlovirus, fuserovirus, geminivirus, hepadnavirus, herpesvirus, hordevirus, hypovirus, ideaovirus, inovirus, iridovirus, levivirus, lipothrix virus, luteovirus, macromovirus, mala Fibovirus (marafivovirus), microvirus, myo (mio) virus, necrovirus, nodavirus, orthomyxovirus, papovavirus, paramyxovirus, partivirus, parvovirus, phycodnavirus, picornavirus, plamavirus (plamavirus) ), podovirus, polydnavirus, potexvirus, potyvirus, poxvirus, reovirus, retrovirus, rhabdovirus, rididivirus, sequevirus (sequevirus), siphovirus, sobemovirus, tectivirus, tenuivirus, It may be selected from the group consisting of Tetravirus, Tobamavirus, Tobravirus, Togavirus, Tombusvirus, Totivirus, Avian virus, Thymovirus, and Ambravirus. In some embodiments, the virus includes deer epidemic hemorrhagic disease virus (EHDV), mouse microvirus (MMV), mouse parvovirus-1 (MPV), cache valley virus. , Vesivirus 2117, porcine circovirus (PCV 1), porcine circovirus 2 (PCV 2), canine parvovirus (CPV), bovine parvovirus (BPV), or bluetongue virus (BTV). Not limited.

  The proteins of the invention are typically active; preferably biologically active proteins. The protein may be any protein of interest for which elimination of any contaminating viral activity is desired.

  The method of the present invention is applicable to all proteins without limitation. As is well known to those of skill in the art, the proteins in a protein-containing sample are typically the deciding factor in the choice of stationary phase and are therefore not a limiting factor in the methods of the invention. The term protein is intended to mean an oligopeptide or polypeptide chain having a secondary structure and optionally a tertiary structure. The term protein includes glycosylated (glycosylated) proteins including oligopeptides, polypeptides, and glycoproteins or proteins linked to nucleic acid sequences. The protein may include any post-translational modifications known to those of skill in the art. A protein may include non-proteinaceous elements, bonds, or modifications. In particular, but not exclusively, with regard to proteins derived from transgenic cells, the proteins of the methods of the invention may comprise one or more unnatural amino acid and/or unnatural isomer.

  The method of the invention is applicable without limitation to proteins from all sources. The protein in the protein-containing sample may be selected from blood protein, milk protein, cellular protein or cell extract protein. The proteins in the protein inclusions are typically blood proteins or proteins from cell extracts, or hormones. More typically, an antibody, or a fragment thereof such as Fab or single chain antibody, a receptor such as a soluble receptor, factor V, factor VII, factor VIII, factor IX, factor X, XI Factors and coagulation factors such as Factor VIII, or growth hormone.

  The terms "polypeptide" and "protein" may be used interchangeably to refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. These terms also refer to amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipid modification (lipidation), acetylation, phosphorylation, or conjugation with labeling moieties, etc. It also includes any other manipulations or modifications of. Also included in the definition are, for example, polypeptides that include analogs of one or more amino acids (including, for example, unnatural amino acids, etc.) and other modifications well known to those of skill in the art.

  The term "antibody" is used in its broadest sense and includes, for example, single monoclonal antibodies (including agonist antibodies, antagonist antibodies, and neutralizing antibodies), antibody compositions with polyepitopic specificity, polyclonal antibodies, single chain. Specifically included are antibodies, multispecific antibodies (eg, bispecific antibodies), immunoadhesins, and fragments of antibodies that exhibit the desired biological or immunological activity. The term "immunoglobulin" (Ig) is used interchangeably herein with antibody.

  The term "antibody fragment" comprises a portion of a full-length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; single chain antibody molecules; diabodies; linear antibodies; and multispecific antibodies formed from antibody fragments. Be done.

  The term “monoclonal antibody” as used herein is obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies that make up the population are identical except for naturally occurring mutations that may be present in small amounts. Is an antibody. In the present specification, a monoclonal antibody includes a part of the heavy chain and/or the light chain which is identical or homologous to a corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass. , While the rest of the chain is derived from another species or is identical or homologous to the corresponding sequence in an antibody belonging to another antibody class or subclass, a “chimeric” antibody, and Fragments of such antibodies that exhibit biological activity are included.

  As mentioned, the exact tailoring of the purification depends on the study of the protein to be purified. In certain embodiments, the separating step of the invention separates the antibody from one or more HCPs. Antibodies that can be successfully purified using the methods described herein include, but are not limited to, human IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4, and IgM antibodies.

  In certain embodiments, the purification strategy of the present invention precludes the use of Protein A affinity chromatography, for example purification of IgG3 antibodies, as IgG3 antibodies bind Protein A inefficiently. Another factor to consider for specific tailoring of the purification scheme is the presence or absence of an Fc region (eg in the context of a full length antibody compared to a Fab fragment), since Protein A binds to the Fc region; Specific germline sequence utilized to generate the antibody of the antibody; and the amino acid composition of the antibody (eg, primary sequence of the antibody and overall charge/hydrophobicity of the molecule), including but not limited to .. Antibodies that share one or more characteristics can be purified using purification strategies tailored to take advantage of the characteristics.

  The protein of the present invention may be a blood protein such as blood coagulation protein, albumin, and/or immunoglobulin. Non-limiting examples of blood proteins include ADAMTS-13, a1-antiplasmin, a2-antiplasmin, antithrombin, antithrombin III, cancer procoagulant (cancer procoagulant), erythropoietin, factor II, factor V. Factor, Factor VI, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, Factor XIII, Fibronectin, Fibrinogen, Heparin cofactor II, High molecular weight kininogen, Intramuscular immunity Globulin, intravenous immunoglobulin, plasminogen, plasminogen activation inhibitor-1, plasminogen activation inhibitor-2, prekallikrein, protein C, protein S, protein Z, protein Z-related protease inhibitor, tissue Factors, tissue plasminogen activator, urokinase, or von Willebrand factor.

  The present invention is suitable for the isolation and purification of antibodies from samples. Typically, the sample comprises a cell line harvest, which cell line is utilized to produce a particular protein, eg, an antibody of the invention.

  Examples 2 and 3, taken together, achieve complete clearance of viruses such as eMuLV by co-purification of two different protein types: recombinant human antibody (IgG1) as one example and Fab as another example. In that respect, it demonstrates that the method of the invention allows protein purification of different types of proteins. In addition, the examples demonstrate co-purification of antibodies by viral inactivation.

  In a preferred embodiment, the sample comprises a protein selected from antibodies or fragments thereof, coagulation factors, and hormones. Antibodies include anti-TNF antibody, anti-IL-6 antibody, anti-IL-8 antibody, anti-IL-12 antibody, anti-IL-16 antibody, anti-IL-20 antibody, anti-IL-21 antibody, Anti-IL-23 antibody, anti-DR3 antibody, anti-CD27 antibody, anti-C5aR-215 antibody, anti-CD30 antibody, anti-TREM-1 antibody, anti-TREM-2 antibody, anti-uPA antibody, anti- uPAR antibody, anti-OX40 antibody, anti-BTLA antibody, anti-GLP-1 antibody, anti-LAIR1 antibody, anti-LAIR2 antibody, anti-LILRA1 antibody, anti-Ly9 antibody, anti-PD1 antibody, anti-Siglec-9 It is preferably selected from the group consisting of antibodies, anti-MMP2 antibodies, anti-MMP6 antibodies, anti-MMP8 antibodies, anti-MMP9 antibodies, and anti-TIM3 antibodies.

  The protein may be a hormone such as a hormone selected from hGH, glucagon, kisspeptin, and insulin.

  In an exemplary embodiment, a resin-filled column is loaded with clarified broth containing the target protein. The column typically contains approximately 1 to 50 g protein per liter resin, for example 5 to 50 g protein per liter resin, typically 5 to 25 g protein per liter resin, more typically resin. Load 10-20 g protein per liter. The column is washed with an aqueous solvent containing about 20-40 mmol/kg sodium caprylate at a pH between about 5-6 and for about 30 minutes after the pH stabilizes. The column is optionally washed with a different solvent to remove impurities and then eluted with a formic acid containing solvent.

  Loading the sample onto the stationary phase may include one or more of the following chromatographic techniques: ion exchange chromatography, affinity chromatography, mixed mode chromatography, and hydrophobic interaction chromatography.

  Thus, the stationary phase may comprise any chromatographic media well known to those skilled in the art, including an inert matrix with immobilized protein binding ligands, proteins, anion exchangers, cation exchangers. Preferably, the stationary phase is a medium for affinity chromatography.

  Purification of proteins from viral contents or inactivation of viruses is the object of the present invention. Example 1 shows that the chromatographic step with a caprylic acid wash on a column with Capto L media completely removes the viral contents, as determined by removal of contaminated eMuLV. .. The complete clearance of eMuLV found in Example 1 is also found in Example 2 on the column with Protein A media. Therefore, protein purification from viral contents or virus inactivation, as determined by clearance of viral load, is independent of column media.

  When using recombinant techniques, the protein may be produced intracellularly, in the periplasm, or directly secreted into the sample medium. In one aspect, such as where the protein is produced intracellularly, prior to loading the sample onto the solid phase, the particulate debris of either host cells or lysed cells (e.g. due to homogenization) is e.g. It may optionally be removed by centrifugation or ultrafiltration. However, the advantage of this method is that such a preliminary step is not essential. When the antibody is secreted into the medium, the supernatant from such an expression system may be first concentrated using a commercially available protein concentration filter.

  When the protein is secreted into the medium, the recombinant host cells can also be separated from the cell culture medium by, for example, tangential flow filtration. The protein of the invention can be purified from the culture medium using the method of the invention.

  In one embodiment, the affinity chromatography step comprises subjecting the sample to a column containing a suitable affinity chromatography carrier. Non-limiting examples of such chromatography carriers include protein A resin, protein L resin, protein G resin, an affinity carrier containing the antigen used to obtain the antibody of interest, and an affinity carrier containing an Fc binding protein. Carriers are included, but are not limited to. Protein A resins are useful for affinity purification and isolation of antibodies such as IgG.

  Following loading of the column, the column may be washed once or multiple times, eg with equilibration buffer. Other washes can be used before eluting the column, including washes with different buffers. The eluate can be monitored using techniques well known to those of skill in the art.

  Suitable cation exchange columns are columns in which the stationary phase contains anionic groups. An example of such a column is the SP Sepharose™ column.

  A suitable mixed mode column is one in which the stationary phase contains mixed mode groups. An example of such a column is the Capto MMC™ column.

  In another embodiment, the sample is subjected to hydrophobic interaction chromatography ("HIC"). Suitable columns are those in which the stationary phase contains hydrophobic groups. An example of such a column is the phenyl Sepharose™ column. The antibody may have formed aggregates during the isolation/purification process. This hydrophobic chromatography step facilitates the elimination of these aggregates. It also helps remove impurities.

  According to the method of the present invention, a protein-containing sample such as cell line medium or plasma is loaded onto a stationary phase so that the target protein is bound to said stationary phase. The method of the invention is not limited to any stationary phase. The examples demonstrate that various stationary phases can be used in the method of the invention. The chromatographic steps can include the following chromatographic techniques: ion exchange chromatography, affinity chromatography, and hydrophobic interaction chromatography. The stationary phase is typically part of a chromatography column. The stationary phase is typically the medium for affinity chromatography. The stationary phase may be selected from ion exchange chromatography columns, high performance protein liquid chromatography, and adsorption fluidized bed (EBA) chromatography columns, preferably ion exchange chromatography columns.

  In one embodiment, the stationary phase comprises an agarose matrix on which is immobilized an immunoglobulin binding protein ligand, such as a recombinant protein or any protein having a strong affinity for the variable region of an antibody kappa light chain. An example of such a stationary phase is a column packed with Capto L medium (GE-Healthcare). Thus, in one embodiment, the stationary phase typically comprises Protein L immobilized on a resin. In a further embodiment, the stationary phase comprises an affinity medium derived from protein A. An example of such a stationary phase is a column packed with Mab Select SuRe medium. In a further embodiment, the stationary phase comprises an agarose matrix with strong cations and/or strong anion exchangers. Examples of such stationary phases include Capto SP ImpRes and Capto Q ImpRes.

  The duration of exposure of a sample to a solution of caprylic acid can vary depending on a number of factors, including but not limited to free caprylic acid concentration, pH, virus type, and temperature. Not done. Table A shows the pH required to obtain the preferred exposure time of the sample to the solution of caprylic acid at various concentrations of caprylate. For very practical purposes, ii. exposing the solid phase to a solution of caprylic acid or exposing the sample to a solution of caprylic acid is less than 2 hours, typically less than 1 hour, for example from 0.05 minutes to It is carried out for 50 minutes, more typically 0.10 minutes to 30 minutes, preferably 0.25 minutes to 15 minutes.

  As can be seen from Tables 6 and 7, viral inactivation is often achieved in less than 5 minutes. However, depending on the conditions (eg, concentration of free caprylic acid, pH, type of virus, and temperature), a maximum of 1 hour, for example, 45 minutes, for example, 30 minutes is practically required.

  In an exemplary embodiment of the invention, the column packed with resin and loaded with target protein is washed with an aqueous solvent containing caprylic acid for about 30 minutes. Prior to eluting the protein with a solvent that removes the target protein, the column is optionally further washed with a different solvent to remove impurities.

  The caprylic acid solution has a pH that inactivates the virus and does not denature the target protein. The caprylic acid solution has a pH that produces nonionic or free caprylic acid. Therefore, the pH of caprylic acid solutions is typically acidic.

  Thus, a solution of caprylic acid has a pH of 7.0 or less, such as a pH between 2 and 7, such as less than 6.5, such as less than 6.1, such as a pH between 2 and 6, such as 2<pH≦6, preferably 3<pH≦. 6, more preferably 4<pH≦6, for example 4.5<pH≦6, most preferably 2<pH<6, preferably 3<pH<6, more preferably 4<pH<6, for example 4.5<pH<6 , For example 4.6≦pH<6, for example about pH 4.6, about pH 4.7, about pH 4.8, about pH 4.9, about pH 5.0, about pH 5.1, about pH 5.2, about pH 5.3, about pH 5.4, about pH 5.5, about pH 5.6, about pH 5.7, about pH 5.8, and about pH 5.9, such as a pH of 4.9<pH<6.

  As can be seen from Example 2, even low concentrations of free caprylic acid are effective in completely inactivating viruses such as eMuLV. In a preferred embodiment of the invention, caprylic acid itself is the buffer (pKa = 4.9). Typically, the caprylic acid solution is 1-100 mM, typically 1-50 mM, including 10-80 mM, e.g. caprylic acid buffer at a concentration of 30-70 mM or 30-50 mM. It is a liquid. Preferably, the concentration of non-ionized caprylic acid is 1-10 mM, such as 2-10 mM, such as 2-8 mM, such as 2-6 mM. In an exemplary embodiment, the buffer used is made by mixing 35 mmol/kg Na caprylate with 4.1 mmol/kg HCl to a pH of about pH 5.6-6.7. The concentration of non-ionized caprylic acid can be determined by the Henderson Hasselbalch equation: pH = pKa + log ([caprylate]/[caprylic acid]).

  Table A shows the pH required at various caprylate concentrations, or conversely at various pH levels, to achieve the minimum concentration of 1 mM free caprylic acid required for protein purification of the invention. It shows various concentrations. In order to inactivate an acceptable amount of virus according to the present invention, the shaded portion has a practically high concentration of free caprylic acid in that the exposure time required for virus inactivation is too long. It represents an embodiment that is too low.

  Typically, the caprylic acid solution has a concentration measured for free caprylic acid of 1 to 500 mM, typically 1 to 50 mM, including 1 to 20 mM, such as 2 to 20 mM or 1-10 mM, more preferably 2-10 mM, for example 2-8 mM, for example 2-6 mM caprylate buffer.

  Typically, the caprylic acid solution is i) caprylate and ii) a pH of less than 7.0, for example 6.5 or less, e.g. pH 1-6.2, and the concentration measured for free caprylic acid is at least 1 mM, For example, it comprises a combination of inorganic or organic acids in proportions to give a solution of caprylic acid of at least 2 mM, for example 2-100 mM, for example 2.5-100 mM.

  Loading of protein-containing samples and/or washing with caprylate buffer can be performed at a variety of temperatures, typically 1-40°C, such as 1-30°C, such as 2-25°C. The low temperature used in Example 4 indicates whether the virus inactivation step is low (2 to 8°C) or relatively high temperature (15 to 25°C) as compared with the temperature used in Example 3. But it shows that it can be achieved.

  Elution of proteins may be performed by conventional techniques such as elution buffers, typically acidic elution buffers with organic acids. In a preferred embodiment, the elution buffer is formic acid. In a preferred embodiment, the elution buffer is prepared by mixing 10 mmol/kg formic acid with 3.4 mmol/kg NaOH to a pH of approximately 3.5. The elution step can also be performed with a buffer solution having a high salt concentration.

  In an exemplary embodiment of the invention, a resin-filled column is loaded with clarified broth containing the target protein; the column is typically loaded with approximately 10-40 g protein per liter resin, e.g. The pH was stable at 2-25° C. and a pH between about 5-6 using an aqueous solvent containing 20-40 g protein/L resin and containing about 20-40 mmol/kg sodium caprylate. Wash the column for about 30 minutes thereafter. The column is optionally further washed with a different solvent to remove impurities and then the protein is eluted with a formic acid containing solvent.

  Further, the present invention is directed to the protein obtained by the method of the present invention in either solid or solution form. In yet another aspect, the invention is directed to one or more pharmaceutical compositions comprising a protein such as an isolated monoclonal antibody or antigen-binding portion thereof obtained by the method of the invention and an acceptable carrier. And

  The following non-limiting examples are provided for illustrative purposes only, to facilitate a more complete understanding of the exemplary embodiments presently discussed. These examples include any of the practices described herein, including those disclosed herein for methods of inactivating viruses that include a lipid coating, and products processed using these methods. It should not be construed as limiting the embodiment.

  While not limiting, the following embodiments are preferred modes of the invention.

1. i. Loading the stationary phase with a protein-containing sample to bind the target protein to the stationary phase;
ii. exposing the solid phase with bound target protein to a solution of caprylic acid, wherein the solution of caprylic acid is at a pH that produces free caprylic acid and the concentration measured for free caprylic acid is between 1 and A step of 50 mM caprylate buffer; and
iii. A method for purifying a target protein from a sample, which comprises a step of eluting the target protein.

2. The method according to embodiment 1, wherein purifying the target protein comprises inactivating viruses in said sample.

3. A method according to embodiment 1 or 2 for preparing a virus-free protein solution.

4. The caprylic acid solution results in i) caprylate and ii) an acidic solution and the concentration measured for free caprylic acid is higher than 1 mM, for example at least 2 mM, for example at least 2.2 mM, typically Comprises a combination with inorganic or organic acids in a proportion that results in at least 2.5 mM caprylic acid.

4. The caprylic acid solution comprises i) caprylate and ii) a pH of less than 7.0, for example pH 6.5 or less, and a concentration measured for free caprylic acid of at least 1 mM, such as at least 2 mM, such as 2-10 mM. A method according to embodiments 1 to 3, comprising a combination with a proportion of an inorganic or organic acid which results in a solution having a concentration of caprylic acid of

5. A group in which the protein comprises an antibody or a fragment thereof, a coagulation factor such as factor V, factor VII, factor VIII, factor IX, factor X, factor XI, and factor VIII, and growth hormone. A method according to embodiments 1 to 4, selected from

6. Embodiments 1-5, wherein the sample is selected from the group consisting of fermentation broth, cell culture, cell line medium, ascites, tissue culture medium, transgenic cell medium, human or animal plasma including plasma fraction Method by any one of.

7. The method according to any one of embodiments 1-6, wherein the stationary phase is a chromatography column.

8. Embodiment 7 wherein the sample is loaded onto a stationary phase selected from ion exchange chromatography columns, affinity chromatography, mixed mode chromatography, high performance protein liquid chromatography, and adsorption fluidized bed (EBA) chromatography columns. By the method.

9. The method according to any one of embodiments 1-8, wherein the caprylic acid solution has a pH that inactivates the virus and does not denature the target protein.

10. The caprylic acid solution has a pH of less than 6.1, such as pH 2-6, such as 2<pH≦6, preferably 3<pH≦6, more preferably 4<pH≦6, such as 4.5<pH≦6, such as 4.6≦pH<6, for example about pH 4.6, about pH 4.7, about pH 4.8, about pH 4.9, about pH 5.0, about pH 5.1, about pH 5.2, about pH 5.3, about pH 5.4, about pH 5.5, about pH 5.6 , About pH 5.7, about pH 5.8, and about pH 5.9, eg, 4.9<pH<6, according to any one of embodiments 1-9.

11. The caprylic acid solution has a concentration measured for free caprylic acid of 1 to 20 mm, such as 2 to 20 mm or 1 to 10 mm, more preferably 2 to 10 mm, such as 2 to 8 mm, such as 2 to 6 A method according to any one of embodiments 1 to 10, which is a caprylic acid buffer solution with a concentration of mm.

12. The method according to any one of embodiments 1-11, wherein the sample comprises an enveloped virus, a non-enveloped virus, or both a non-enveloped virus and an enveloped virus.

13. The method according to any one of embodiments 1-12, which is automated or is part of an automated protein purification process.

14. The method according to any one of embodiments 1-13, further inactivating mycoplasma.

15. A method for purifying a protein selected from coagulation factors, antibodies, or Fabs thereof, comprising:
i. loading a sample containing one or more of the above proteins onto a stationary phase for affinity chromatography;
ii. exposing the solid phase to a solution of caprylic acid at a pH of 4.9<pH<6.2 at a concentration measured for free caprylic acid of 2-10 mM, for example 2-8 mM, for example 2-6 mM, and exposing the virus to Inactivating and washing away;
iii. A method comprising the step of eluting the target protein.

  A further advantage of the method of the present invention is that it provides a virus inactivated sample, or a virus-free sample, and also a mycoplasma inactivated sample. Further, the method of the invention provides a sample with a reduced amount of impurities such as host cell derived protein (HCP).

[Example 1]
Cell line media containing recombinant human Fab fragments was 0.22 μm filtered and then contaminated with eMuLV virus. The mixed medium was applied to a column packed with Capto L medium (GE-Healthcare). After loading, the column was first washed with equilibration buffer (20 mM phosphate, 150 mM NaCl, pH 7.2) and then with 35 mM caprylate buffer at pH 5.5 (5.8 mM free caprylic acid). After a 30 minute wash with caprylate buffer, the Fab fragment was eluted from the column with 20 mM formic acid, pH 3.5. The process temperature was 15-25°C. The yield of Fab fragment was 97%, and eMuLV was not detected in the elution fraction containing Fab fragment (Table 1).

A chromatographic step with a caprylic acid wash on a column with Capto L media shows complete removal of contaminated eMuLV with an LRV greater than 6.0 log ₁₀ .

[Example 2]
The cell line medium containing the recombinant human antibody (IgG ₁ ) was filtered with a 0.22 μm filter to incorporate eMuLV virus. The contaminated medium was loaded onto a column packed with Protein A (Mab Select SuRe, Ge Healthcare). After loading, the column was first washed with equilibration buffer (50 mmol/kg phosphate, 300 mmol/kg NaCl, pH 7.0) and then 32 mmol/kg caprylate buffer (2.9 mM free caprylic acid) at pH 5.8. Washed with. Low concentrations of caprylate and high pH were used to demonstrate the effectiveness of this process in the worst conditions (low caprylic acid concentration). After the pH had stabilized, the wash was continued for 30 minutes. After three additional washes (equilibration buffer, 50 mmol/kg phosphate, 1 mol/kg NaCl, and then equilibration buffer), the mAb was eluted from the column with formate buffer, pH 3.5. The yield of mAb was 100%, and eMuLV was not detected in the elution fraction containing mAb (Table 2). The process temperature was 15-25°C.

  The complete clearance of eMuLV seen in Example 1 is also seen in the column with Protein A medium. Therefore, the clearance of the incorporated load is independent of the medium in the column. In addition, even low concentrations of free caprylic acid are effective for complete clearance of eMuLV.

[Example 3]
Cell line medium containing recombinant human antibody (IgG ₄ ) was filtered through a 0.22 μm filter to incorporate eMuLV virus. The contaminated medium was loaded onto a column packed with protein A (Mab Select SuRe, Ge Healthcare). After loading, the column was first washed with equilibration buffer (50 mmol/kg phosphate, 300 mmol/kg NaCl, pH 7.0) and then 32 mmol/kg caprylate buffer (2.9 mM free caprylic acid) at pH 5.8. Washed with. After the pH had stabilized, the wash was continued for 30 minutes. After three additional washes (equilibration buffer, 50 mmol/kg phosphate, 1 mol/kg NaCl) and then equilibration buffer), the mAb was eluted from the column with formate buffer, pH 3.5. The yield of mAb was 90%, and eMuLV was not detected in the elution fraction containing mAb (Table 3). The process temperature was 15-25°C.

  This example using a human antibody separate from Example 2 shows that the complete clearance of eMuLV is independent of the monoclonal antibody in the cell line medium.

[Example 4]
Cell line medium containing recombinant human antibody (IgG ₁ ) was filtered through a 0.22 μm filter to incorporate eMuLV virus. The contaminated medium was loaded onto a column packed with Protein A (Mab Select SuRe, Ge Healthcare). After loading, the column was first washed with equilibration buffer (50 mmol/kg phosphate, 300 mmol/kg NaCl, pH 7.0) and then 35 mmol/kg caprylate buffer (3.9 mM free caprylic acid) at pH 5.7. Washed with. After the pH had stabilized, the wash was continued for 30 minutes. After three additional washes (equilibration buffer, 50 mmol/kg phosphate, 1 mol/kg NaCl, and then equilibration buffer), the mAb was eluted from the column with formate buffer, pH 3.5.

  The yield of mAb was 90%, and eMuLV was not detected in the elution fraction containing mAb (Table 4). The process temperature was 2-8°C.

  The lower temperatures used in this example compared to the temperatures used in Example 3 indicate that complete clearance of eMuLV is achieved both at low temperatures (2-8°C) and above. Be done.

[Example 5]
170 liters of cell line medium containing recombinant human antibody (IgG ₁ ) was bisected in a pilot plant and then separately, protein A (Mab Select SuRe, from an automated chromatography system (AKTA pilot, GE Healthcare). Ge Healthcare).

  One was loaded onto a Protein A column (7.1 liter Mab Select SuRe, Ge Healthcare) and the column was then loaded with 3 buffers (equilibration buffer, (50 mmol/kg phosphate, 1 mol/kg NaCl), and then equilibration buffer. Liquid). The mAb was eluted from the column using formate buffer, pH 3.5. The eluate was manually processed at low pH (3.7) for 60 minutes.

  The other was treated as in Example 4, but with a programmed caprylic acid wash step during protein A chromatography (7.1 liter Mab Select SuRe, Ge Healthcare) to treat the eluate at low pH. Not included.

  The eluate was then loaded separately onto a cation exchange column (2.2 liter Poros 50 HS, Life Technologies). After washing the column with the equilibration buffer (37.4 mM acetate buffer, pH 5), the column was eluted with a linear gradient of 0-300 mM NaCl in acetate buffer, pH 5.

  The concentration of HMWP was about 2% in both eluates, but increased to 3% after low pH treatment (see table). After cation exchange, the concentration of HMWP was about 2% in both the low pH treated and the caprylic acid wash during protein A chromatography. The concentration of CHO-HCP was only about half in the eluate from the protein A column washed with caprylic acid compared to the concentration in the low pH treated eluate (see table). After cation exchange, the concentration of CHO-HCP in those treated with a caprylic acid wash during protein A chromatography remained at half the amount of CHO-HCP treated in low pH.

  The method with a caprylic acid wash during protein A chromatography was about 45 minutes faster than the method with low pH treatment of the eluate from protein A. The yield of monoclonal antibody was almost the same in the two parts after cation exchange (Table 5).

  The examples show that lower concentrations of CHO-HCP are achieved by using a caprylic acid wash step during chromatography on a column with protein A. Lower concentrations of CHO-HCP are still visible after the next chromatographic step (cation exchange). It also avoids an increase in HMWP after the low pH step. Using the method with a caprylic acid wash saved 45 minutes of process time compared to the method with a low pH step.

[Example 6]
The inactivation of MuLV at different caprylic acid concentrations and different pH was investigated (Table 6). Complete inactivation of MuLV is achieved after 5 minutes when increasing the pH and increasing the caprylic acid concentration so that the free caprylic acid concentration is maintained at least about 4 mM in the pH range of 5.0-6.0. Was done. A caprylic acid concentration of approximately 2 mM increased the reaction time, resulting in partial inactivation after 30 minutes for these most difficult viruses. No inactivation was observed below 1 mM.

  The concentration of caprylic acid is pH dependent and only free caprylic acid inactivates the enveloped virus. Therefore, the efficiency of 60 mM caprylic acid was further investigated in the pH range of pH 5.9-6.3. Complete inactivation of MuLV over 5.4 log10 was seen within 5 minutes over this pH range (Table 7). At pH 6.3, the caprylic acid concentration is 2.2 mM, which is also valid in this test.

  At a total concentration of caprylic acid of 60 mM, MuLV inactivation was highly effective at pH ranging from pH 5.9 to 6.2. Best results were obtained when the pH was maintained below 6.1 with 60 mM caprylic acid.

  It has been shown that to be effective, the concentration of free caprylic acid should be at least 1 mM, preferably at least about 2 mM, such as about 2.2 mM, such as at least about 2.5 mM, or at least 2.8 mM.

As one of ordinary skill in the art will readily appreciate, high concentrations of other salts such as CaCl ₂ or NaCl tend to reduce the solubility of caprylic acid. As one of ordinary skill in the art will readily appreciate, concentrations of non-caprylate higher than 500 mM, such as higher than 100 mM or higher than 50 mM can affect the ability of free caprylic acid to be produced. It should be avoided as it has potential. However, one skilled in the art will readily recognize that the concentration of caprylic acid should not be higher than its solubility.

  A protein purification method involving virus inactivation with caprylic acid was shown to be highly effective and rapid for inactivating general enveloped viruses, including MuLV virus, at free caprylic acid concentrations above 1 mM. Was done. While particular features of the present invention have been illustrated and described herein, one of ordinary skill in the art will appreciate that many modifications, substitutions, changes and equivalents may be made. Therefore, it is to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims (14)

i. Loading the stationary phase with a protein-containing sample to bind the target protein to the stationary phase;
ii. exposing the stationary phase with the bound target protein to a solution of caprylic acid, wherein the solution of caprylic acid is at a pH that produces free caprylic acid and the concentration measured for free caprylic acid is 1 A caprylate buffer that is ~50 mM;
iii. A method for purifying the target protein from a sample, comprising the step of eluting the target protein, comprising :
The method wherein the stationary phase is a chromatography column .   The method of claim 1, wherein purifying the target protein comprises inactivating viruses in the sample.   3. The method according to claim 1 or 2 for preparing a virus-free protein solution.   The caprylic acid solution comprises i) caprylate and ii) a proportion of an inorganic or organic acid which results in a solution of caprylic acid at a concentration of at least 1 mM, measured at a pH below 7.0, of free caprylic acid. 4. A method according to any one of claims 1 to 3 including a combination with.   The method according to any one of claims 1 to 4, wherein the protein is selected from the group consisting of an antibody or a fragment thereof, a coagulation factor, and growth hormone.   6. The sample of claim 1-5, wherein the sample is selected from the group consisting of fermentation broth, cell culture, cell line medium, ascites, tissue culture medium, transgenic cell medium, human or animal plasma including plasma fraction. The method according to any one of items. The sample is loaded on a stationary phase selected from ion exchange chromatography columns, affinity chromatography columns, mixed mode chromatography columns, high performance protein liquid chromatography columns, and adsorption fluidized bed (EBA) chromatography columns. Item 7. The method according to any one of Items 1 to 6 . The caprylic acid solution, virus inactivation, and the has a pH that does not denature the target protein, the method according to any one of claims 1-7. The caprylic acid solution has a pH of pH 6.1 or less, the method according to any one of claims 1-8. The caprylic acid solution, caprylic acid buffer concentrations 1 to 20 mM as measured for free caprylic acid method according to any one of claims 1-9. 11. The method of any one of claims 1-10 , wherein the sample comprises an enveloped virus, a non-enveloped virus, or both a non-enveloped virus and an enveloped virus. The method according to any one of claims 1 to 12 , which is automated or is part of an automated protein purification process. Further inactivating mycoplasma method according to any one of claims 1 to 12. A method for purifying a protein selected from coagulation factors, antibodies, or Fabs thereof, comprising:
i. loading a sample containing one or more of the above proteins onto a stationary phase for affinity chromatography;
ii. exposing the stationary phase to a solution of caprylic acid having a concentration of 2-10 mM measured for free caprylic acid and a pH of 4.9<pH<6.2 to inactivate and wash away the virus;
iii. A method comprising the step of eluting the target protein.